In proteins, the extent of synchrotron radiolysis is dependent on the solvent accessibility of the reactive residues. Since the solvent accessibility of discrete residues will be altered upon interaction of Ha protein with ligand, synchrotron radiolysis provides a method to probe these changes and, in combination with mass spectrometric analysis, identify the amino acid residues involved in ligand binding. HA change in the hydroxyl radical induced modification of the protein Hversus the protein-ligand complex is representative of a footprint Hfor residues that interact at the binding site. We have chosen to Hstudy the interaction between human platelet profilin (HPP) and Hpoly-L-proline as a model protein-ligand complex, as its crystal structure has elucidated the specific amino acid residues in HPP that bind poly-L-proline and that the structure of HPP is not altered substantially by the binding of this ligand. The poly-L-proline peptide interacts strongly with HPP as indicated by the dissociation constants for the complex . Residues Trp3 and Tyr6 in the N-terminal Lys-C peptide of HPP participate in binding as does Trp31. The fraction of unmodified protein that remains after millisecond x-ray exposure is determined by mass spectrometry and is shown as a function Hof time together with data for the Lys-C peptides that contain the Hbinding residues after proteolysis. These are compared with data from Hradiolysis of the protein-ligand complex to examine changes to the Htime-resolved modification of protein with the incorporation of Hligand.